The aim of this project is to engineer a toolbox of easy-to-use, highly specific and targetable modifying factors that can manipulate the epigenome in a predictable manner. The promoters of tumor suppressor genes are hypermethylated and inactive in cancer cells. Epigenomes favoring survival in the presence of a drug, or ectopic expression of an oncogene, render chemotherapeutics ineffective and tumor cells to grow. A better understanding of cancer epigenetics is important for cancer research and therapy. However, our understanding of the functional consequences of these modifications is still far from complete and our ability to engineer specific modifications in a targeted manner is in its infancy. Drugs affecting epigenetic information are in clinical use for cancer, but cause broad changes in gene expression due to lack of specificity. Limited success has been achieved in attaching epigenetic enzymes to programmable DNA-binding domain such as zinc fingers (ZFs) or TALEs. Because of the ease in cloning guide RNAs and due to a high binding specificity, there is much interest in using the far more useful CRISPR/Cas9 system to modify the epigenome. However, of the ~20 epigenetic modifiers reported to date only two have used the CRISPR/Cas9 system. This has not been due to lack of interest or effort; it more likely reflects an unresolved problem encountered when employing the CRISPR/Cas9 system. We hypothesize that important structural features of dCas9 targeted to DNA are different than for ZFs or TALEs, and that a change in past design strategies is required to create CRISPR epigenetic modifiers. We feel that this is an important problem that needs to be solved so that the CRISPR/Cas9 system can be used to alter the epigenome in a site-specific manner. Aim 1 of this project is to design CRISPR/Cas9-based systems that can alter specific epigenetic marks. We hypothesize that epigenetic writers and erasers that directly modify DNA are inhibited by structural features of dCas9 bound to DNA. To circumvent this problem, we will use alternative design strategies to tether epigenetic modifying complexes to dCas9, and measure their ability to alter specific marks. In Aim 2, we will characterize how CRISPR/Cas9 epigenetic tools alter gene expression. We hypothesize that longer regions of epigenetic modification should lead to a graded increase in regulation, and greater persistence. We will assess the length of modification needed to achieve robust gene regulation, determine if the factors provide a graded or threshold response, and examine how long the new epigenetic and gene expression patterns persist. Finally, we will examine genome-wide changes in epigenetic marks and gene expression. The tools and information developed in this proposal will form a foundation from which numerous applications in tumorigenesis, drug resistance, and new cancer therapeutics could be developed.